Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep red. Eye-catching examples of carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. All photosynthetic organisms, as well as some bacteria and fungi, synthesize carotenoids. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage. For example, animals do not have the ability to synthesize carotenoids but must instead obtain these nutritionally important compounds through their dietary sources.
Industrially, only a few carotenoids are used for food colors, animal feeds, pharmaceuticals, and cosmetics, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol. 70:181–191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis, but only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. As a result, carotenoids produced from these plants are very expensive.
Structurally, the most common carotenoids are 40-carbon (C40) terpenoids; however, carotenoids with only 30 carbon atoms (C30; diapocarotenoids) are detected in some species. Biosynthesis of carotenoids is derived from the isoprene biosynthetic pathway and its five-carbon universal isoprene building block, isopentenyl pyrophosphate (IPP). This biosynthetic pathway can be divided into two portions: 1) the upper isoprene pathway, which leads to the formation of farnesyl pyrophosphate (FPP); and 2) the lower carotenoid biosynthetic pathway, comprising various crt genes which convert FPP into long C30 and C40 carotenogenic compounds characterized by a long central chain of conjugated double bonds. Both portions of this pathway are shown in FIG. 1.
The degree of the carbon backbone's unsaturation, conjugation, isomerization and functionalization determines the specific carotenoids' unique absorption characteristics and colors. This variation in properties is the result of a suite of crt genes, such as the crtE, crtX, crtY, crtI, crtB, crtZ, crtW, crtO, crtR, crtM, crtN and cdtN2 genes shown in FIG. 1. Additionally, various other crt genes are known that enable the intramolecular conversion of linear C30 and C40 compounds to produce numerous other functionalized carotenoid compounds by: (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/glycosylation, or any combination of these processes.
The genetics of C40 carotenoid pigment biosynthesis has been extremely well studied in the Gram-negative pigmented bacteria of the genera Pantoea, formerly known as Erwinia. In both E. herbicola EHO-10 (ATCC 39368) and E. uredovora 20D3 (ATCC 19321), the crt genes are clustered in two genetic units, cdt Z and crt EXYIB (U.S. Pat. No. 5,656,472; U.S. Pat. No. 5,545,816; U.S. Pat. No. 5,530,189; U.S. Pat. No. 5,530,188; and U.S. Pat. No. 5,429,939) These genes have subsequently been sequenced and identified in a suite of other species of bacterial, fungal, plant and animal origin. Several reviews discuss the genetics of carotenoid pigment biosynthesis, such as those of G. Armstrong (J. Bact. 176: 4795–4802 (1994); Annu. Rev. Microbiol. 51:629–659 (1997)).
The abundant knowledge concerning the genetics of C40 biosynthesis has permitted production of a number of natural C40 carotenoids from genetically engineered microbial sources. Examples include:                1.) Lycopene (Farmer, W. R. and Liao, J. C., Biotechnol. Prog. 17: 57–61(2001); Wang, C. et al., Biotechnol Prog. 16: 922–926 (2000); Misawa, N. and Shimada, H., J. Biotechnol. 59:169–181 (1998); Shimada, H. et al. Appl. Environ. Microbiol. 64:2676–2680 (1998));        2.) β-carotene (Albrecht, M. et al., Biotechnol. Lett. 21: 791–795 (1999); Miura, Y. et al., Appl. Environ. Microbiol. 64:1226–1229 (1998); U.S. Pat. No. 5,691,190);        3.) Zeaxanthin (Albrecht, M. et al., supra); Miura, Y. et al., supra); and        4.) Astaxanthin (U.S. Pat Nos. 5,466,599; 6,015,684; 5,182,208; and U.S. Pat. No. 5,972,642).Further, genes encoding various elements of the lower C40 carotenoid biosynthetic pathway have been cloned and expressed in various microbes (e.g., U.S. Pat. Nos. 5,656,472; 5,545,816; 5,530,189; 5,530,188; 5,429,939; and U.S. Pat. No. 6,124,113).        
Despite abundant knowledge and understanding of the C40 carotenoid pathway, C30 pigment biosynthesis is both less well-understood and less prevalent in nature. Early studies by Kleinig, H. et al. (Z. Naturforsch 34c: 181–185 (1979); Z. Naturforsch 37c: 758–760 (1982)) examined the structure and biosynthesis of C30 carotenoic acid glucosyl esters produced in Pseudomonas rhodos (subsequently renamed Methylobacterium rhodinum) by mutational analysis. To date, presence of diapocarotenoids has been discovered in Streptococcus faecium (Taylor, R. F. and Davies, B. H., J. Biochem. 139:751–760 (1974)), M. rhodinum (Kleinig, H. et al., supra; Taylor, R. F. Microbiol. Rev. 48:181–198 (1984)), genera of the photosynthetic heliobacteria (Takaichi, S. et al., Arch. Microbiol. 168: 277–281 (1997)), and Staphylococcus aureus (Marshall, J. H. and Wilmoth, G. J., J. Bacteriol. 147:900–913 (1981)). All appear to have a diapophytoene precursor, from which all subsequent C30 compounds are produced.
The relevant genes responsible for C30 carotenoid pigment biosynthesis are known to include crtM and crtN in Staphylococcus aureus. The diapophytoene desaturase CrtN can function to some extent in the C40 pathway, and the phytoene desaturase CrtI of the C40 carotenoids can also function in the C30 pathway (Raisig and Sandmann, Biochim. Biophys. Acta 1533:164–170 (2001)). Microbial genomic sequencing effort revealed several ORFs in other organisms with significant homology to crtM or crtN of S. aureus (Xiong et al., Proc. Natl. Acad. Sci. USA 95:14851–14856 (1998);Takami et al., Nucleic Acids Res, 30:3927–3935 (2002)). However, their roles in C30 carotenoid synthesis have not been determined. Investigators J. H. Marshall and G. J. Wilmonth (J. Bacteriol. 147:914–919 (1981)) suggested that mixed-function oxidases are responsible for the introduction of oxygen functions to produce the aldehyde and carboxylic acid of 4,4-diaponeurosporene. However, none of the genes responsible for the addition of functionality to the terminal methyl group of the linear C30 carotenoid molecule have been identified despite characterization of the resulting carotenoids. Methods for industrial production of C30 carotenoids are lacking. It would be desirable to develop methods to produce C30 carotenoids (and specifically, C30-aldehyde carotenoids such as diaponeurosporene monoaldehyde, diapocarotene monoaldehyde, and diapocarotene dialdehyde (shown in FIG. 2) to increase the number of carotenoids industrially available for use in food colors, animal feeds, pharmaceuticals, and cosmetics. Additionally, the presence of aldehyde group(s) within the C30-aldehyde carotenoids also provides active “handles” useful for cross-linking or chemical modification of the carotenoids to facilitate desired applications.
The microbial production of C30-aldehyde carotenoids to a significant level has not been previously reported and is especially problematic due to the toxicity of aldehydes to bacterial systems (see, for example: Ingram et al., Biotechnol. Bioeng. 65:24–33 (1999); Marnett et al., Proc. Natl. Acad. Sci. USA 94:8652–8657 (1997); Mee and O'Donovan, Mutagenesis 8:577–581 (1993); Kawazoe et al., Mutat. Res. 156:153–161(1985)). In light of these needs, the problem to be solved is to develop a system for production of C30-aldehyde carotenoids.
Applicants have solved the stated problem by engineering microorganisms for the production of C30-aldehyde carotenoids. Specifically, Applicants have identified two unique open reading frames encoding the enzymes CrtN and CrtN2 from a Methylomonas sp. and co-expressed these enzymes with the CrtM and CrtN C30-carotenoid biosynthesis enzymes from Staphylococcus aureus in Escherichia coli. This leads to the production of diapocarotene dialdehyde. Subsequent metabolic engineering of the host demonstrated that synthesis of this C30-carotenoid could be modified such that it would be produced in levels suitable for industrial purposes.